The term "tPA" means "tissue plasminogen activator". The term "PAI" means plasminogen activator inhibitor. "PAI 1" is plasminogen activator inhibitor one and is sometimes called endothelial plasminogen activator inhibitor. "PAI 2" is plasminogen activator inhibitor two and is sometimes called placental plasminogen activator inhibitor. The term ".alpha..sub.2 AP" means alpha 2 antiplasmin and is a protein found in blood of normal individuals that inhibits the enzyme plasmin. The term "fibrinogen digests" includes the products from digestion of fibrinogen or fibrin with proteolytic enzymes, such as plasmin.
Investigation of tissue plasminogen activator inactivation in plasma has been hampered by poor methodology. A specific and sensitive method for measuring tPA in plasma samples where potential fibrinolytic inhibitors were neutralized by controlled acidification was described. (See Wiman, B., et al., Clin. Chim Acta, 127, 279-288, 1982). tPA subsequently measured by this method exhibited a parabolic rate assay. (See R.ang.nby, M., et al, Thromb. Res. 27, 743-749, 1982). With this method it was also possible to specifically determine inhibitory activity to tPA in plasma and kinetic evidence for a fast tPA inhibitor in plasma was presented.
Assuming the formation of a stoichiometric 1:1 complex, a rate constant of about 10.sup.7 M.sup.-1 .multidot.s.sup.-1 was calculated, and the plasma concentration of the new inhibitor in healthy individuals was determined as 8.+-.2 unit/ml (1 unit=inhibition of 1 international unit of tPA). The tPA inhibitory content was also determined in plasma from various patients. High inhibitory activity content was frequently found in patients with deep venous thrombosis, hemostatic problems during late pregnancy, or severe coronary heart disease. (See Chmielewska, J., et al., Thromb. Res. 31, 427-436 (1983)). The PAI activity observed is now known to be the result of PAI1 activity.
There are several assay systems that are commercially available for the measurement of tPA and PAI which utilize native tPA. However, the results concerning PAI levels obtained from these assays are not always reliable because of the cleavage of tPA into the two chain form of the protein. Native one-chain tPA is cleaved by plasmin or by trypsin after the Arg in the sequence -Gln-Phe-Arg-Ile-Lys- in the tPA protein. This creates a problem when trying to measure the PAI1 level in a biological fluid by inhibition of the tPA activity because two chain tPA also reacts rapidly with PAI 2 and reacts much faster than single chain tPA with other protease inhibitors such as .alpha..sub.2 AP.
Thus, what is needed is a tPA that is resistant to cleavage by proteolytic enzymes to prevent formation of two chain tPA in the preparation procedure or during the assay procedure. With such a tPA, levels of PAI1 activity could be more accurately measured.
Another problem encountered in the prior art methods of measuring tPA and
is the fact that tPA and PAI1 activity are unstable after blood is collected and the activity of the two proteins decreases after blood is collected. In blood with high PAI1 levels, the tPA activity can decrease by 50% in about one minute. The PAI1 activity typically has a half life of 4 hours at room temperature.
When tPA is assayed in blood, it has been found that polylysine is an effective stimulator of tPA activity. However, it has also been found that polylysine is not an effective activator of tPA in biological fluids other than blood and blood plasma. Thus, what is needed is to identify the preparation in plasma that, together with polylysine, constitutes the effective tPA stimulator which will allow one to design better methods to determine the tPA level. The improved methods will allow one to perform tPA activity assays in non-plasma systems.
Another problem encountered in the prior art is that plasma samples must be acidified and incubated for relatively long periods of time at low pH to destroy the plasmin inhibitory activity in the plasma sample that interferes with the assay. What is needed is to identify the inhibitory activity and neutralize this with specific antibodies. This will make both tPA activity and PAI activity more convenient to assay.
Yet another problem encountered in the prior art of determining tPA activity is that the tPA activity is underestimated in biological fluids that also contain PAI1 activity. This is because PAI1 in this study is found to be relatively stable during acidification and will react with tPA when the sample is neutralized during the tPA assay procedure. What is needed is a method of inhibiting PAI1 activity in the assay system when measuring tPA activity in biological fluids.